Rotavirus infection is the greatest cause of diarrhea related deaths in infants and young children. Every year rotavirus gastroenteritis causes the deaths of 310,000-590,000 infants and young children, worldwide. International health agencies have promoted the development of rotavirus vaccine as the best method for the prevention of morbidity and mortality associated with rotavirus infection. In 1997 and, again, in 2000, the WHO recommended that all new rotavirus vaccines should be tested in Asia and Africa and that this testing should be performed concurrently with trials conducted in the United States and Europe. By doing this safety and efficacy of vaccines might be demonstrated in poor, developing countries early during development, thereby accelerating the availability of new vaccines to the children who are most in need of them.
All rotavirus vaccines developed to date have been based on live rotavirus strains that have been isolated from humans or animals and in vitro reassorted and adapted to cell culture, formulated for oral delivery. Both monovalent and multivalent animal based strains have demonstrated efficacy as candidate vaccines. RotaShield (Wyeth-Ayerst) was licensed but then was withdrawn.
The human rotavirus strain 116E, natural human-bovine reassortant, naturally attenuated
is characterized as a human G9 strain into which a single bovine VP4 gene, homologous to P[11] gene segment is naturally introduced. The I321, strain is characterized as a G10P [11] is composed primarily of bovine genes and has only two gene segments, VP5 and VP7 of human origin. These two rotavirus vaccine strains individually have been prepared as pilot lots of monovalent oral rotavirus vaccine liquid formulations for clinical trials to be conducted in India.
Bharat Biotech International Ltd. (BBIL) obtained the human rotavirus strains, 116E and I321 from National Institute of Health (NIH) under the material transfer agreement with National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, USA. The complete genomic sequence of rotavirus strains 116E and I321 is reported. The original 116E (G9[P11]) and I321 (G10P[11]) were adapted to grow in cell culture by passages in primary African green monkey kidney (AGMK) cells then in MA104 cell substrate and later in Serially Passaged AGMK (SPAGMK). MA104 and SPAGMK cell substrates are not approved by National Regulatory Authorities (NRA) for commercial vaccine production. Hence it is preferable to adapt 116E and I321 and other rotavirus vaccine strains to approved, certified, licensed and fully characterized cell substrate like Vero cell substrate and/or human diploid cells like MRC-5.
WO 02/11540 describes rotavirus vaccine formulations which include buffering agents appropriate for oral administration of rotavirus vaccines. The formulations disclosed in WO 02/11540 also include compounds to stabilize the vaccine compositions against potency loss. More specifically, the compositions disclosed in WO 02/11540 comprise a sugar, phosphate and at least one carboxylate. The stabilities achieved with the formulations of WO 02/11540 vary greatly, and especially at temperatures over 20 degrees Celsius appear to show considerable losses in potency. Accordingly, it was an object of the present invention to provide for alternative compositions of a viral antigen, preferably compositions of rotavirus, which show a good stability.
The objects of the present invention are solved by a composition comprising a viral antigen, a first protein different from said viral antigen, said first protein being selected from human serum albumin (HSA) and recombinant human albumin (rHA), a second protein different from said viral antigen, which second protein is at least partially hydrolyzed, said second protein being selected from lactalbumin. The second protein confers enhanced stability to the vaccine formulation than either of the protein alone.
In one embodiment the composition further comprises three different disaccharides, wherein, preferably, said three different disaccharides are selected from sucrose, lactose, maltose, trehalose, cellobiose, gentobiose, melibiose and turanose. Where as sucrose is used as primary sugar and secondary sugar is selected from the combination of sucrose, lactose, maltose and trehalose. The combination of sugars at particular concentrations further confers stability to the vaccine formulation containing protein additives. The term “primary sugar”, as used herein, is meant to refer to a sugar that is present in a composition with other sugars, at an amount greater than of any of the other sugars present. Such other sugars are herein also referred to as “secondary sugars”. The “primary sugar” may also be referred to herein as “bulk sugar”.
In one embodiment said second protein which is at least partially hydrolyzed is selected from lactalbumin hydrolyzate, yeast hydrolyzate, peptone, and gelatin hydrolyzate.
In one embodiment said first protein is present in the formulation in an amount in the range of from 0.1% (w/v) to 2% (w/v), preferably 0.1% (w/v) to 0.45 (w/v) and said second protein which is at least partially hydrolyzed is present in the formulation, as at least partial hydrolyzate, in an amount in the range of from 0.01% (w/v) to 10% (w/v).
Preferably, said three different disaccharides, or said primary sugar and said at least two secondary sugars are one of the following combinations:                a) sucrose, lactose and maltose,        b) sucrose, maltose and trehalose,        c) sucrose, lactose and trehalose,        d) maltose, lactose and trehalose.        
Preferably, the amount of said three different disaccharides together in the formulation is from 20% (w/v) to 70% (w/v).
In a preferred embodiment the amount of sucrose, if present, is from 40% (w/v) to 55% (w/v), preferably 50% (w/v), the amount of lactose, if present, is from 0.1% (w/v) to 10.0% (w/v), preferably 0.5% (w/v), the amount of maltose, if present, is from 0.1% (w/v) to 10.0% (w/v), preferably 0.5% (w/v), and the amount of trehalose, if present, is from 0.1% (w/v) to 10.0% (w/v), preferably 0.5% (w/v).
In one embodiment said viral antigen is a live virus, such as a live attenuated virus, wherein, preferably, said live virus is selected from the group comprising rotaviruses.
Preferably, said live virus is a human live virus, such as human rotavirus.
In a particularly preferred embodiment said human rotavirus is rotavirus strain 116E or I321.
In one embodiment, the composition according to the present invention comprises a live rotavirus at a titre in the range of from 104 to 107 FFU/0.5 ml, human serum albumin in the range of from 0.1% (w/v) to 0.45% (w/v), lactalbumin hydrolysate in the range of from 0.01% (w/v) to 10% (w/v), and either
a) sucrose at an amount in the range of from 40% to 55% (w/v), and
lactose at an amount in the range of from 0.1% to 10.0% (w/v), and
maltose at an amount in the range of from 0.1% to 10.0% (w/v),
or
b) sucrose at an amount in the range of from 40% to 55% (w/v), and
maltose at an amount in the range of from 0.1% to 10.0% (w/v), and
trehalose at an amount in the range of from 0.1% to 10.0% (w/v).
In one embodiment the composition according to the present invention further comprises a buffer to adjust the pH of said composition to a value in the range of from 6.8 to 7.8, wherein, preferably, said buffer is a phosphate/citrate buffer.
In one embodiment the composition according to the present invention is made up in Eagles Minimum essential medium, wherein, preferably, it is buffered in a phosphate/citrate buffer at a pH between 6.8 and 7.8.
Preferably, said phosphate/citrate buffer is approximately 310 mM phosphate and approximately 100 mM citrate.
In one embodiment the composition according to the present invention is a vaccine.
The preparation of stable vaccine formulation presents significant challenges. The interactions between the incorporated excipients in the vaccine composition determine the formulation stability. Protein and sugar hydrogen bonding need to be dominant to result with stabilization of the vaccine. The effective contacts of sugar and antigenic protein vaccine should be with appropriate ratio to keep the vaccine stable. A critical sugar concentration is required to have number of protein and sugar hydrogen bonding to keep the vaccine stable for a given period of time, at a given temperature. Sugars have the ability to hydrogen bond to phospholipids membrane and proteins by substituting for structural water. The present study provides the stabilizers used to stabilize the live attenuated Rota virus 116 E and I321 against 2-8° C., 25° C. and 37° C. for an extended period of time.
Appropriate combination of the sugars and proteins with the buffering component preserve and allow the survival and activity of the virus on a long storage period. These additives give structural support to the suspended Rota Virus in the liquid state. Trehalose is preferred to use as stabilizer in vaccine formulation, since it stabilizes the protein structure of the virus and restore the potency on long storage. Combination of Sucrose and Maltose makes effective contacts of sugar and protein (vaccine) to keep the vaccine stable. Human serum albumin supported by Lactalbumin hydrolyzate play prominent role in effective contact of sugar and vaccine protein to keep the vaccine stable.
The objects of the present invention are also solved by the use of a viral antigen, a first protein and a second protein, each being as defined above, and, optionally, three different disaccharides, as defined above, for the manufacture of a composition according to the present invention, preferably for the manufacture of a vaccine, for the treatment or prevention of virus associated, preferably rotavirus associated diseases, such as diarrhea and gastroenteritis.
Preferably, said treatment or prevention comprises administering three oral doses of a safe and effective amount of the composition to an infant within 8-20 weeks of age at the time of dose 1.
Preferably, the use according to the present invention is for the prevention of a virus infection, preferably a rotavirus infection and/or rotavirus gastroenteritis in humans.
The objects of the present invention are also solved by a method of treatment or prevention of virus associated, preferably rotavirus associated diseases in humans by administering to a human an effective amount of the composition according to the present invention.
Preferably, the method according to the present invention, comprises administering three oral doses of a safe and effective amount of the composition to an infant within 8-20 weeks of age at the time of dose 1.
In one embodiment the method according to the present invention, for the prevention of a virus infection, preferably a rotavirus infection and/or rotavirus gastroenteritis in humans.
The objects of the present invention are solved by a method of adapting a virus to a suitable cell line, such as Vero cells, comprising serially passaging said virus through cultures of said suitable cell line, each passage occurring in a medium in the presence of calcium chloride and trypsin.
Preferably, said calcium chloride is present in the range of from 0.1 mg/ml to 1 mg/ml, and said trypsin is present in the range of from 0.1 μg/ml to 30 μg/ml.
In one embodiment said serial passages comprise 2-20 passages, preferably 2-5 passages.
Preferably, each passage occurs over a time period in the range of from 24 hours to 10 days.
Preferably, said virus is human rotavirus.
The present inventors have found that by including a first protein selected from human serum albumin, recombinant human albumin and a second protein which is at least partially hydrolyzed, in a pharmaceutical composition, the stability of this composition with respect to its viral potency can be enhanced. Especially at temperatures 25° C. and 37° C., the compositions according to the present invention show an enhanced stability.
The phrase “the second protein is at least partially hydrolyzed”, as used herein, is meant to refer to a scenario, in which this second protein has been at least partially been broken down into its respective amino acid building blocks. The aforementioned phrase is therefore also meant to include scenarios, wherein the second protein does not exist as a complete molecule anymore, but only as a collection of fragments thereof. The aforementioned phrase is meant to also include a scenario wherein the second protein is fully hydrolyzed. All these scenarios are also meant to be included by the phrase “protein hydrolyzate”, such as “lactalbumin hydrolyzate”, which may include a fully hydrolyzed protein, i.e. a protein broken down into its respective amino acids, or a protein partially broken down, such that a collection of peptides and amino acids exist. Such protein hydrolyzates can be easily made by someone skilled in the art, for example by acid hydrolysis, or they can be commercially obtained.
The stabilizing effect achieved by the presence of the first protein and the second protein is optionally enhanced by the presence of a combination of three different disaccharides, or of a combination of a primary sugar and at least two secondary sugars. Preferred combinations are sucrose, lactose and maltose, and sucrose, maltose and trehalose. The compositions according to the present invention may be used as a vaccine for vaccination against virus infection and virus associated diseases. Preferably, these compositions are buffered at an appropriate pH, usually between 6 and 8, preferably between 6.8 and 7.8. For example, in one embodiment, the composition according to the present invention may be formulated in Eagles minimum essential medium. Preferably, this composition is buffered, for example using a phosphate/citrate buffer.
The present inventors have also devised a method of adapting a virus to a suitable cell-line, which method may be for example used with rotavirus. Such method is performed by serially passaging the virus through cell cultures, wherein the passaging occurs in the presence of calcium chloride and trypsin. This allows for an easy way of adapting virus to a given cell line and furthermore enables the production of virus at high titers.
More specifically and by way of example, the present inventors have adapted 116E and I321 rotavirus strains to Vero cells to produce high titer rotavirus harvest and further characterized the adapted 116E and I321 for making stable, live, monovalent, liquid rotavirus vaccine compositions. Prior to vaccination, oral antacid composition of citrate-bicarbonate buffer is given to buffer stomach acidity of the child.
All rotavirus vaccine strains reported till date are either natural, live, human bovine, naturally or artificially attenuated rotavirus strains or genetically engineered human, bovine strains with various combinations of VP4, VP7 and other genes of human, bovine rotavirus strains. The rotavirus strains 116E (G9P[11]) and I321 (G10P[11]) are natural human-bovine reassortant, naturally attenuated and confer substantial level of immunity in infants and young children. Also the ability of 116E strain to replicate in newborns without causing disease in presence of high titers of maternal antibody make it more promising live, naturally attenuated monovalent rotavirus vaccine candidate.
The present invention provides for pharmaceutical compositions of virus, preferably rotavirus, which show high stability and longevity especially at temperatures above 20° C. The present invention also provides a method for adapting rotavirus, e.g. natural human-bovine reassortant, naturally attenuated rotavirus strains 116E (G9P [11]) and I321 (G10P[11]) to suitable cells, e.g. Vero cells. The method include optimized dose of trypsin (0.1 μg/ml to 30 μg/ml) and/or calcium chloride (100 μg/ml to 1000 μg/ml) for virus activation and virus maintenance medium where high titer (104 to 108 FFU/ml) of virus harvest is within one to ten days. Also use of the adapted strains for making stable, live, monovalent, liquid rotavirus vaccine composition is envisaged. Furthermore, the present invention provides for the use of a viral antigen, a first protein, a second protein and, optionally, a combination of three different disaccharides for the manufacture of a composition according to the present invention for the treatment or prevention of virus associated diseases, preferably rotavirus associated diseases.